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Risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil
Preventive Veterinary Medicine 78 (2007) 286–295 www.elsevier.com/locate/prevetmed
Risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil Jean Carlos Ramos Silva a,c,*, Maria Fernanda Vianna Marvulo b, Ricardo Augusto Dias b, Fernando Ferreira b, Marcos Amaku b, Cristina Harumi Adania c, Jose´ Soares Ferreira Neto b a
Departamento de Medicina Veterina´ria (DMV), Universidade Federal Rural de Pernambuco (UFRPE), Av. Dom Manoel de Medeiros, s/n, Dois Irma˜os, Recife, PE, Cep. 50171-900, Brazil b Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal (VPS), Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), Universidade de Sa˜o Paulo (USP), Av. Prof. Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, SP, Cep. 05508-270, Brazil c Centro Brasileiro Para Conservac¸a˜o dos Felinos Neotropicais, Associac¸a˜o Mata Ciliar, Caixa Postal 2020, Jundiaı´, SP, Cep. 13212-010, Brazil Received 28 October 2004; received in revised form 23 October 2006; accepted 24 October 2006
Abstract From September 1995 to February 2001, blood samples were collected from 865 neotropical felids belonging to 8 different species. These animals were housed in 86 institutions located in 78 cities of 20 Brazilian states. Our goal was to identify the risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil. All serum samples were tested by the modified agglutination test (MAT), using formalin-fixed whole tachyzoites and mercaptoethanol. For each animal an individual questionnaire was filled with questions about tattoo number, felid species, age, sex, origin, number of animals in the group, introduction of new animals in the group, time in the institution, eating meat previously frozen for a period <7 days in the last 6 months, eating meat of run-over or euthanized animals in the last 6 months, predation of rodents or birds in the last 6 months and presence of domestic cats near the enclosures in the last 6 months. The total sero-prevalence was 55% (95% CI: 52%, 57%). We estimated a prevalence of 46% (95% CI: 40%, 54%) for jaguarundi (Puma yagouaroundi); 58% (95% CI: 53%, 63%) for ocelot (Leopardus pardalis); 50% (95% CI: * Corresponding author at: Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, 52171-900, Dois Irma˜os, Recife, PE, Brazil. Tel.: +55 81 3320 6429; fax +55 81 3320 6400. E-mail address: [email protected] (J.C.R. Silva). 0167-5877/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2006.10.013
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45%, 56%) for oncilla (L. tigrinus); 54% (95% CI: 46%, 62%) for margay (L. wiedii); 12% (95% CI: 4%, 31%) for Pampas-cat (L. colocolo); 83% (95% CI: 65%, 93%) for Geoffroy’s-cat (L. geoffroyi); 64% (95% CI: 50%, 68%) for jaguar (Panthera onca) and 48% (95% CI: 42%, 54%) for puma (Puma concolor). Multiple logistic regression was used to evaluate the association between the variables in the questionnaire and sero-positivity to T. gondii. We concluded that the independent risk factors for toxoplasmosis were: age >3 years (OR = 4.75 [2.75; 8.2]), eating meat previously frozen for a period <7 days (OR = 2.23 [1.24; 4.01]), and consumption of animals that were run-over or euthanized (OR = 1.64; [1.14; 2.37]). # 2006 Elsevier B.V. All rights reserved. Keywords: Toxoplasma gondii; Sero-prevalence; Risk factors; Captive neotropical felids; Zoological garden; Brazil
1. Introduction Neotropical felids are represented by 10 species with a range extending from Mexico to Argentina, except for puma (Puma concolor), ocelot (Leopardus pardalis) and jaguarundi (Puma yagouaroundi), which may also be found in United States of America and Canada. Brazil hosts eight of these species (Oliveira, 1994) and only jaguarundi is not endangered (Fonseca et al., 1994). Nowadays, the greatest cause of decrease in felid population in the wild is the loss of habitats (Oliveira, 1994). Many species of wild felids are kept in zoological parks and private breeding centers in Brazil, as part of a national conservationist programme. Studies on neotropical felids, both in captivity and in the wild, will enable a better understanding of the epidemiology of zoonotic and nonzoonotic transmissible diseases (Munson and Cook, 1993; Thrusfield, 1995; Silva et al., 2001a). Felids are important in the dissemination of Toxoplasma gondii infection to humans and other animals worldwide, because they are the only animals that excrete oocysts in the environment (Frenkel et al., 1970; Miller et al., 1972; Dubey and Beattie, 1988). Also, there are only a few reports on T. gondii infection in wild felids in Brazil, both in zoos (Sogorb et al., 1977; Silva et al., 2001a,b) and in animals in the wild (Ferraroni and Marzochi, 1980; Ferraroni et al., 1980). Toxoplasmosis is a common infection that rarely evolves to clinical disease. Australian marsupials and neotropical primates are the groups most susceptible to clinical and fatal toxoplasmosis (Dubey and Beattie, 1988; Garrel, 1999). Toxoplasmosis is a serious threat to endangered primates in Brazil, as described by Epiphanio et al. (2000) in zoos in the state of Sa˜o Paulo. That report showed fatal outbreaks of the disease in golden-headed lion tamarins (Leontopithecus chrysomelas) and emperor marmosets (Saguinus imperator). Serological studies have shown a high prevalence of antibodies anti-T. gondii in zoo animals that indicates an important spread of this agent in this environment (Riemann et al., 1974; Sogorb et al., 1977; Ippen et al., 1981; Gorman et al., 1986; Choi et al., 1987; Murata, 1989; Zhang et al., 2000; Silva et al., 2001a,b; Spencer et al., 2003; Kikuchi et al., 2004). In studies of toxoplasmosis sero-prevalence performed in felids in zoos and breeding centers, the possible risk factors suggested included the presence of domestic cats roaming in the
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zoo (Gorman et al., 1986; Murata, 1989; Lukesˇova´ and Litera´k, 1998), the introduction of wild felids into the group and the predation of birds and small rodents (Riemann et al., 1974; Ippen et al., 1981; Dubey et al., 1988). Possible routes of transmission were: ingestion of raw meat from horses (Riemann et al., 1974; Gorman et al., 1986; Spencer et al., 2003), bovine foetuses and cattle (Van Rensburg and Silkstone, 1984; Spencer et al., 2003), swine (Dorny and Fransen, 1989), deer (Stover et al., 1990; Lappin et al., 1991; Spencer et al., 2003) and rabbits (Lukesˇova´ and Litera´k, 1998). However, those authors only speculated; no formal study was performed to identify risk factors associated with felid infection by T. gondii. Our objective was to identify risk factors associated with sero-positivity for T. gondii in neotropical felids kept in captivity in Brazil.
2. Material and methods Between September 1995 and February 2001, 71 zoological parks and 15 breeding centers in Brazil were visited (Fig. 1). These institutions corresponded to all zoological parks and breeding centers registered at the Brazilian Institute of Environment and Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renova´veis, IBAMA) that housed captive felids until February 2001. All felids in each enclosure (n = 540) were sampled, totalling 865 animals from 8 species. Each felid was sampled just once. Individuals were anesthetized using ketamine (Francotar, Francodex; Vetaset, Fort Dodge; 10–12 mg/kg i.m.) and xylazine (Rompum, Bayer; 1–2 mg/kg i.m.) or tiletamine and zolazepan (Zoletil, Virbac; 7–10 mg/kg i.m.). Blood samples were collected from a jugular, brachial, safena, or femoral veins using vacutainer tubes or syringes, stored for 4– 6 h at approximately 4 8C, and then centrifuged. Sera were stored at 20 8C until analysis. All sera were transported frozen and analyzed for the presence antibodies to T. gondii at the Parasite Biology, Epidemiology and Systematics Laboratory of USDA, Beltsville, MD, by means of the modified agglutination test (MAT) using formalin-fixed whole tachyzoites and 2-mercapthoethanol (Dubey and Desmonts, 1987; Dubey and Thulliez, 1989). Cut-off value was titer 20 (Silva et al., 2001a). For each animal an individual questionnaire, with 12 questions, was filled out with: (1) IBAMA tattoo number; (2) felid species; (3) age (years); (4) sex; (5) origin (wild, born in the institution, another institution, donation or circus); (6) social group (alone, couple or group 3); (7) introduction of new animals in the group (yes/no); (8) time in the institution (months); (9) eating meat previously frozen for a period <7 days in the last 6 months (yes/ no); (10) eating meat of run-over or euthanized animals in the last 6 months (yes/no); (11) predation of rodents or birds in the last 6 months (yes/no); (12) presence of domestic cats near the enclosures in the last 6 months (yes/no). All the questionnaires were administered by the first author together with another veterinary trained and supervised by him. The possibility of oocyst ingestion by these wild felids was assessed by means of questions on the presence of cats around the enclosures, on animal being kept isolated or in groups and on the introduction of felids in the group (questions 6, 7 and 12). The possibility of ingestion of viable tissue cysts in the meat was assessed through questions about the
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Fig. 1. Brazilian institutions registered at IBAMA, visited for the collection of blood samples from neotropical felids, 1995–2001.
origin of the meat offered to the animals or predation of prey by agile felids (questions 10 and 11), and on the use of previous freezing of the meat (question 9). Meat storage at 12 8C, for at least 5 days, destroys T. gondii tissue cysts (Kotula et al., 1991). The sex, age, origin and time of captivity of the animals were also analyzed. Data on the questionnaires and serological tests were analyzed using commercial software (SPSS, 1999). Total sero-prevalence was calculated, as well as sero-prevalences by animal species, considering each enclosure as a cluster. In the study of the risk factors, a univariable analysis was performed using the Chisquare (x2) test, and those variables that presented P < 0.10 were offered to multiple logistic regression. The multiple analysis was then performed, using the stepwise forward
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method (Hosmer and Lemeshow, 1989). We used the Hosmer and Lemeshow goodness-offit test to assess the model fit. We examined the variance-inflation factor (Kleinbaum et al., 1998) to test for evidence of collinearity between the variables that passed screening but were not selected for the model and the risk factors in the final model.
3. Results and discussion We choose the MAT because it does not need a species-specific conjugate and presents better sensitivity for cats than the Sabin–Feldman dye test, ELISA, indirect haemagglutination and latex haemagglutination tests (Dubey and Thulliez, 1989; Dubey et al., 1995). We certainly had misclassifications of the serum-status because MAT is an indirect diagnostic test and its sensitivity and specificity were established only for pigs (S.E. = 85.7%, SP = 94.6%, Gamble et al., 2005). However, many authors used MAT test for surveys in domestic cats (Silva et al., 2002; Gauss et al., 2003; Dubey et al., 2004b, 2006; Pena et al., 2006) and wild felids (Aramini et al., 1998; Zhang et al., 2000; Labelle et al., 2001; Silva et al., 2001a,b; Dubey et al., 2004a; Mucker et al., 2006), usually considering titers 20 or 25 as cut-offs. Sero-prevalences are shown in Table 1. In spite of having tested all neotropical felids present in all institutions registered at the IBAMA, we calculated the confidence intervals Table 1 Sero-prevalences to Toxoplasma gondii (MAT titer 20) in 865 captive neotropical felids from Brazil (1995– 2001) Species
Positives
Sampled
%
95% CI
Geoffroy’s-cat Leopardus geoffroyi Jaguar Panthera onca Jaguarundi Puma yagouaroundi Margay Leopardus wiedii Ocelot Leopardus pardalis Oncilla Leopardus tigrinus Pampas-cat Leopardus colocolo Puma Puma concolor
10 135 46 34 97 66 1 83
12 212 99 63 168 131 8 172
83 64 46 54 58 50 12 48
65, 93 50, 68 40, 54 46, 62 53, 63 45, 56 4, 31 42, 54
Total
472
865
55
52, 57
Table 2 Reports on the occurrence of antibodies anti-Toxoplasma gondii in captive neotropical felids Species Jaguarundi, Geoffroy’s-cat, ocelot, puma Jaguarundi, ocelot, puma Ocelot, oncilla Puma a
Sampled
% Positive
Test a
Country
Reference
6
11
54
IFAT
USA
Spencer et al. (2003)
9 11 29
11 15 92
81 73 31
ELISA DT LAT
USA Brazil Americas
Lappin et al. (1991) Sogorb et al. (1977) Kikuchi et al. (2004)
Positives
DT, Sabin–Feldman dye test; ELISA, enzyme-linked immunoabsorbent assay; IFAT, indirect fluorescent antibody test; LAT, latex agglutination test.
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Table 3 Distribution of neotropical felids per enclosure in zoos and breeding centers from Brazil (1995–2001) Number of animals per enclosure
% of enclosures % of animals in this condition Sero-prevalence (%)
1
2
3
4
5
7
9
58 36 55
30 37 56
8 16 51
3 7 48
0.6 2 33
0.2 1 86
0.2 1 89
assuming that this population is dynamic and that there are other institutions and also persons, not registered at the IBAMA, that kept these species in captivity. The most important reports on the occurrence of antibodies anti-Toxoplasma gondii in captive neotropical felids are summarized in Table 2. These data show that the infection is widely disseminated among neotropical felids kept in captivity in the Americas and suggest that the risk of exposure to T. gondii in these animals varies greatly from place to place. The total sero-prevalences for males and females were, respectively, 55% (95% CI: 52%, 58%) and 54% (95% CI: 51%, 57%), showing that there were no differences of susceptibility or risk of exposure between sexes. Table 3 shows that most of the animals were kept alone or in couples or groups of two animals of the same sex. It was not possible to carry out a logistic regression with random effects, technically the most suitable method in this case, because the computational algorithms did not converge due to the fact that many clusters had only one animal (Table 3). Thus, to estimate the risk factors, we used a logistic regression without random effects. Table 4 Distribution of risk factors for sero-positivity to T. gondii in 865 captive wild felids from 86 Brazilian zoos and breeding centers (1995–2001) Variable
Sero-positive
Sero-negative
Exposed
Total
Exposed
Total
Age >3 years
440
464
282
397
Animal origin Wild Born in the institution Another institution Donation Circus
126 81 92 46 3
464 494 464 464 464
97 112 72 30 1
368 398 398 398 398
Animal lived in a group Another animal was introduced in the group Time in the institution >1 year Eating meat previously frozen for a period <7 days in the last 6 months Eating meat of run-over or euthanized animals in the last 6 months Predation of rodents and birds in the last 6 months Presence of domestic cats near the enclosures in the last 6 months
279 136 270 386 271 90 248
426 458 320 439 442 442 442
238 91 183 295 160 57 192
366 397 270 378 383 383 383
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Table 5 Results of the multiple logistic regression analysis for risk factors associated with sero-positivity to T. gondii in 865 captive wild felids from 86 zoos and breeding centers from Brazil (1995–2001) Variable
b
S.E.(b)
OR
95% CI
P*
Age >3 years Eating meat previously frozen for a period <7 days Eating meat of run-over or euthanized animals
1.56 0.80 0.49
0.28 0.29 0.19
4.75 2.23 1.64
2.75, 8.2 1.24, 4.01 1.14, 2.37
0.001 0.007 0.008
Intercept = 2.12, P < 0.001; log likelihood statistic = 701.82; Hosmer–Lemeshow x2 = 1.075 (d.f. = 3, P = 0.78). * Wald’s test.
Table 4 shows the results of the univariable analysis and Table 5 shows the final model of the multiple logistic regression. The observed significance level for the Hosmer and Lemeshow goodness-of-fit Chisquare (x2 = 1.075, d.f. = 3) was 0.78, so the logistic regression model fitted the data reasonably well. Examining the variance-inflation factor (all values below 1.2), no collinearity was observed between the variables that passed screening but were not selected for the model and the risk factors in the final logistic model. It is reasonable to suppose that the greater the age, the greater the possibility of exposure to the risk of infection by T. gondii. However, the possibility of intervention on the variable ‘‘age’’, in a zoo, is null for the animals already housed. Knowledge that older cats are more likely to be infected might be used when planning management to other felids and animal keepers. It is interesting to us observe that although some variables related to the risk of exposure by means of the ingestion of oocysts were selected and submitted to multiple logistic regression (Table 4), none of them remained in the final model, indicating that the greatest risk of exposure is the ingestion of viable cysts in tissues. This is related to feed management, a variable in which intervention is possible. Thus, in the zoo, the exposure of neotropical felids to the T. gondii could be reduced by feeding the animals only with meat previously frozen at 12 8C for a period >7 days (Kotula et al., 1991). In Brazil, feeding captive felids with carcasses of runover or euthanized animals is an acceptable and very common practice. The implementation of this routine depends on the availability of equipment and storage capacity, which are possible to be arranged, but are not always found in some visited institutions. Considering that the elimination of oocysts has been shown in 5 out of 10 existing neotropical felids species (Jewell et al., 1972; Miller et al., 1972; Pizzi et al., 1978; Aramini et al., 1998; Lukesˇova´ and Litera´k, 1998), it is possible to state that after the intervention, a reduction in the load of oocysts eliminated in the environment is expected. The reduction of the oocysts environmental contamination is particularly interesting when felids are kept near by Australian marsupials and neotropical primates, which are species highly susceptible to toxoplasmosis (Dubey and Beattie, 1988; Garrel, 1999; Epiphanio et al., 2000).
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4. Conclusions The most effective way to reduce the risk of exposure of captive neotropical felids to T. gondii infection in Brazil and consequently reduce the oocysts environmental contamination is feeding these animals only with meat previously frozen at 12 8C for a period >7 days.
Acknowledgements The authors are grateful to the zoos and breeding centers staffs, IBAMA and Associac¸a˜o Mata Ciliar. They also thank Dr. J.P. Dubey (ARS, USDA) for laboratory support and CAPES and FAPESP for financial support.
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Silva, J.C.R., Gennari, S.M., Ragozo, A.M., Amajones, V.R., Magnabosco, C., Yai, L.E., Ferreira-Neto, J.S., Dubey, J.P., 2002. Prevalence of Toxoplasma gondii antibodies in sera of domestic cats from Guarulhos and Sa˜o Paulo, Brazil. J. Parasitol. 88, 419–420. Sogorb, F., Jamra, L.F., Guimara˜es, E.C., 1977. Toxoplasmose em animais de Sa˜o Paulo, Brasil. Rev. Inst. Med. Trop. Sa˜o Paulo 19, 191–194. Spencer, J.A., Higginbotham, M.J., Blagburn, B.L., 2003. Seroprevalence of Neospora caninum and Toxoplasma gondii in captive and free-ranging nondomestic felids in the United States. J. Zoo Wildl. Med. 34, 246–249. SPSS Inc., 1999. Spss Base 9.0. SPSS Inc., Chicago. Stover, J., Jacobson, E.R., Lukas, J., Lappin, M.R., Buergelt, C.D., 1990. Toxoplasma gondii in a collection of nondomestic ruminants. J. Zoo Wildl. Med. 21, 295–301. Thrusfield, M., 1995. Veterinary Epidemiology, second ed., vol. 148. Blackwell Science, Oxford, pp. 191–198. Van Rensburg, I.B.J., Silkstone, M.A., 1984. Concomitant feline infectious peritonitis and toxoplasmosis in a cheetah (Acinonyx jubatus). J. South Afr. Vet. Assoc. 55, 205–207. Zhang, S.Y., Wei, M.X., Zhou, Z.Y., Yu, J.Y., Shi, X.Q., 2000. Prevalence of antibodies to Toxoplasma gondii in the sera of rare wildlife in the Shangai Zoological Garden, People’s Republic of China. Parasitol. Int. 49, 171–174.
Risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil Jean Carlos Ramos Silva a,c,*, Maria Fernanda Vianna Marvulo b, Ricardo Augusto Dias b, Fernando Ferreira b, Marcos Amaku b, Cristina Harumi Adania c, Jose´ Soares Ferreira Neto b a
Departamento de Medicina Veterina´ria (DMV), Universidade Federal Rural de Pernambuco (UFRPE), Av. Dom Manoel de Medeiros, s/n, Dois Irma˜os, Recife, PE, Cep. 50171-900, Brazil b Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal (VPS), Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), Universidade de Sa˜o Paulo (USP), Av. Prof. Orlando Marques de Paiva, 87, Cidade Universita´ria, Sa˜o Paulo, SP, Cep. 05508-270, Brazil c Centro Brasileiro Para Conservac¸a˜o dos Felinos Neotropicais, Associac¸a˜o Mata Ciliar, Caixa Postal 2020, Jundiaı´, SP, Cep. 13212-010, Brazil Received 28 October 2004; received in revised form 23 October 2006; accepted 24 October 2006
Abstract From September 1995 to February 2001, blood samples were collected from 865 neotropical felids belonging to 8 different species. These animals were housed in 86 institutions located in 78 cities of 20 Brazilian states. Our goal was to identify the risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil. All serum samples were tested by the modified agglutination test (MAT), using formalin-fixed whole tachyzoites and mercaptoethanol. For each animal an individual questionnaire was filled with questions about tattoo number, felid species, age, sex, origin, number of animals in the group, introduction of new animals in the group, time in the institution, eating meat previously frozen for a period <7 days in the last 6 months, eating meat of run-over or euthanized animals in the last 6 months, predation of rodents or birds in the last 6 months and presence of domestic cats near the enclosures in the last 6 months. The total sero-prevalence was 55% (95% CI: 52%, 57%). We estimated a prevalence of 46% (95% CI: 40%, 54%) for jaguarundi (Puma yagouaroundi); 58% (95% CI: 53%, 63%) for ocelot (Leopardus pardalis); 50% (95% CI: * Corresponding author at: Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, 52171-900, Dois Irma˜os, Recife, PE, Brazil. Tel.: +55 81 3320 6429; fax +55 81 3320 6400. E-mail address: [email protected] (J.C.R. Silva). 0167-5877/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2006.10.013
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45%, 56%) for oncilla (L. tigrinus); 54% (95% CI: 46%, 62%) for margay (L. wiedii); 12% (95% CI: 4%, 31%) for Pampas-cat (L. colocolo); 83% (95% CI: 65%, 93%) for Geoffroy’s-cat (L. geoffroyi); 64% (95% CI: 50%, 68%) for jaguar (Panthera onca) and 48% (95% CI: 42%, 54%) for puma (Puma concolor). Multiple logistic regression was used to evaluate the association between the variables in the questionnaire and sero-positivity to T. gondii. We concluded that the independent risk factors for toxoplasmosis were: age >3 years (OR = 4.75 [2.75; 8.2]), eating meat previously frozen for a period <7 days (OR = 2.23 [1.24; 4.01]), and consumption of animals that were run-over or euthanized (OR = 1.64; [1.14; 2.37]). # 2006 Elsevier B.V. All rights reserved. Keywords: Toxoplasma gondii; Sero-prevalence; Risk factors; Captive neotropical felids; Zoological garden; Brazil
1. Introduction Neotropical felids are represented by 10 species with a range extending from Mexico to Argentina, except for puma (Puma concolor), ocelot (Leopardus pardalis) and jaguarundi (Puma yagouaroundi), which may also be found in United States of America and Canada. Brazil hosts eight of these species (Oliveira, 1994) and only jaguarundi is not endangered (Fonseca et al., 1994). Nowadays, the greatest cause of decrease in felid population in the wild is the loss of habitats (Oliveira, 1994). Many species of wild felids are kept in zoological parks and private breeding centers in Brazil, as part of a national conservationist programme. Studies on neotropical felids, both in captivity and in the wild, will enable a better understanding of the epidemiology of zoonotic and nonzoonotic transmissible diseases (Munson and Cook, 1993; Thrusfield, 1995; Silva et al., 2001a). Felids are important in the dissemination of Toxoplasma gondii infection to humans and other animals worldwide, because they are the only animals that excrete oocysts in the environment (Frenkel et al., 1970; Miller et al., 1972; Dubey and Beattie, 1988). Also, there are only a few reports on T. gondii infection in wild felids in Brazil, both in zoos (Sogorb et al., 1977; Silva et al., 2001a,b) and in animals in the wild (Ferraroni and Marzochi, 1980; Ferraroni et al., 1980). Toxoplasmosis is a common infection that rarely evolves to clinical disease. Australian marsupials and neotropical primates are the groups most susceptible to clinical and fatal toxoplasmosis (Dubey and Beattie, 1988; Garrel, 1999). Toxoplasmosis is a serious threat to endangered primates in Brazil, as described by Epiphanio et al. (2000) in zoos in the state of Sa˜o Paulo. That report showed fatal outbreaks of the disease in golden-headed lion tamarins (Leontopithecus chrysomelas) and emperor marmosets (Saguinus imperator). Serological studies have shown a high prevalence of antibodies anti-T. gondii in zoo animals that indicates an important spread of this agent in this environment (Riemann et al., 1974; Sogorb et al., 1977; Ippen et al., 1981; Gorman et al., 1986; Choi et al., 1987; Murata, 1989; Zhang et al., 2000; Silva et al., 2001a,b; Spencer et al., 2003; Kikuchi et al., 2004). In studies of toxoplasmosis sero-prevalence performed in felids in zoos and breeding centers, the possible risk factors suggested included the presence of domestic cats roaming in the
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zoo (Gorman et al., 1986; Murata, 1989; Lukesˇova´ and Litera´k, 1998), the introduction of wild felids into the group and the predation of birds and small rodents (Riemann et al., 1974; Ippen et al., 1981; Dubey et al., 1988). Possible routes of transmission were: ingestion of raw meat from horses (Riemann et al., 1974; Gorman et al., 1986; Spencer et al., 2003), bovine foetuses and cattle (Van Rensburg and Silkstone, 1984; Spencer et al., 2003), swine (Dorny and Fransen, 1989), deer (Stover et al., 1990; Lappin et al., 1991; Spencer et al., 2003) and rabbits (Lukesˇova´ and Litera´k, 1998). However, those authors only speculated; no formal study was performed to identify risk factors associated with felid infection by T. gondii. Our objective was to identify risk factors associated with sero-positivity for T. gondii in neotropical felids kept in captivity in Brazil.
2. Material and methods Between September 1995 and February 2001, 71 zoological parks and 15 breeding centers in Brazil were visited (Fig. 1). These institutions corresponded to all zoological parks and breeding centers registered at the Brazilian Institute of Environment and Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renova´veis, IBAMA) that housed captive felids until February 2001. All felids in each enclosure (n = 540) were sampled, totalling 865 animals from 8 species. Each felid was sampled just once. Individuals were anesthetized using ketamine (Francotar, Francodex; Vetaset, Fort Dodge; 10–12 mg/kg i.m.) and xylazine (Rompum, Bayer; 1–2 mg/kg i.m.) or tiletamine and zolazepan (Zoletil, Virbac; 7–10 mg/kg i.m.). Blood samples were collected from a jugular, brachial, safena, or femoral veins using vacutainer tubes or syringes, stored for 4– 6 h at approximately 4 8C, and then centrifuged. Sera were stored at 20 8C until analysis. All sera were transported frozen and analyzed for the presence antibodies to T. gondii at the Parasite Biology, Epidemiology and Systematics Laboratory of USDA, Beltsville, MD, by means of the modified agglutination test (MAT) using formalin-fixed whole tachyzoites and 2-mercapthoethanol (Dubey and Desmonts, 1987; Dubey and Thulliez, 1989). Cut-off value was titer 20 (Silva et al., 2001a). For each animal an individual questionnaire, with 12 questions, was filled out with: (1) IBAMA tattoo number; (2) felid species; (3) age (years); (4) sex; (5) origin (wild, born in the institution, another institution, donation or circus); (6) social group (alone, couple or group 3); (7) introduction of new animals in the group (yes/no); (8) time in the institution (months); (9) eating meat previously frozen for a period <7 days in the last 6 months (yes/ no); (10) eating meat of run-over or euthanized animals in the last 6 months (yes/no); (11) predation of rodents or birds in the last 6 months (yes/no); (12) presence of domestic cats near the enclosures in the last 6 months (yes/no). All the questionnaires were administered by the first author together with another veterinary trained and supervised by him. The possibility of oocyst ingestion by these wild felids was assessed by means of questions on the presence of cats around the enclosures, on animal being kept isolated or in groups and on the introduction of felids in the group (questions 6, 7 and 12). The possibility of ingestion of viable tissue cysts in the meat was assessed through questions about the
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Fig. 1. Brazilian institutions registered at IBAMA, visited for the collection of blood samples from neotropical felids, 1995–2001.
origin of the meat offered to the animals or predation of prey by agile felids (questions 10 and 11), and on the use of previous freezing of the meat (question 9). Meat storage at 12 8C, for at least 5 days, destroys T. gondii tissue cysts (Kotula et al., 1991). The sex, age, origin and time of captivity of the animals were also analyzed. Data on the questionnaires and serological tests were analyzed using commercial software (SPSS, 1999). Total sero-prevalence was calculated, as well as sero-prevalences by animal species, considering each enclosure as a cluster. In the study of the risk factors, a univariable analysis was performed using the Chisquare (x2) test, and those variables that presented P < 0.10 were offered to multiple logistic regression. The multiple analysis was then performed, using the stepwise forward
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method (Hosmer and Lemeshow, 1989). We used the Hosmer and Lemeshow goodness-offit test to assess the model fit. We examined the variance-inflation factor (Kleinbaum et al., 1998) to test for evidence of collinearity between the variables that passed screening but were not selected for the model and the risk factors in the final model.
3. Results and discussion We choose the MAT because it does not need a species-specific conjugate and presents better sensitivity for cats than the Sabin–Feldman dye test, ELISA, indirect haemagglutination and latex haemagglutination tests (Dubey and Thulliez, 1989; Dubey et al., 1995). We certainly had misclassifications of the serum-status because MAT is an indirect diagnostic test and its sensitivity and specificity were established only for pigs (S.E. = 85.7%, SP = 94.6%, Gamble et al., 2005). However, many authors used MAT test for surveys in domestic cats (Silva et al., 2002; Gauss et al., 2003; Dubey et al., 2004b, 2006; Pena et al., 2006) and wild felids (Aramini et al., 1998; Zhang et al., 2000; Labelle et al., 2001; Silva et al., 2001a,b; Dubey et al., 2004a; Mucker et al., 2006), usually considering titers 20 or 25 as cut-offs. Sero-prevalences are shown in Table 1. In spite of having tested all neotropical felids present in all institutions registered at the IBAMA, we calculated the confidence intervals Table 1 Sero-prevalences to Toxoplasma gondii (MAT titer 20) in 865 captive neotropical felids from Brazil (1995– 2001) Species
Positives
Sampled
%
95% CI
Geoffroy’s-cat Leopardus geoffroyi Jaguar Panthera onca Jaguarundi Puma yagouaroundi Margay Leopardus wiedii Ocelot Leopardus pardalis Oncilla Leopardus tigrinus Pampas-cat Leopardus colocolo Puma Puma concolor
10 135 46 34 97 66 1 83
12 212 99 63 168 131 8 172
83 64 46 54 58 50 12 48
65, 93 50, 68 40, 54 46, 62 53, 63 45, 56 4, 31 42, 54
Total
472
865
55
52, 57
Table 2 Reports on the occurrence of antibodies anti-Toxoplasma gondii in captive neotropical felids Species Jaguarundi, Geoffroy’s-cat, ocelot, puma Jaguarundi, ocelot, puma Ocelot, oncilla Puma a
Sampled
% Positive
Test a
Country
Reference
6
11
54
IFAT
USA
Spencer et al. (2003)
9 11 29
11 15 92
81 73 31
ELISA DT LAT
USA Brazil Americas
Lappin et al. (1991) Sogorb et al. (1977) Kikuchi et al. (2004)
Positives
DT, Sabin–Feldman dye test; ELISA, enzyme-linked immunoabsorbent assay; IFAT, indirect fluorescent antibody test; LAT, latex agglutination test.
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Table 3 Distribution of neotropical felids per enclosure in zoos and breeding centers from Brazil (1995–2001) Number of animals per enclosure
% of enclosures % of animals in this condition Sero-prevalence (%)
1
2
3
4
5
7
9
58 36 55
30 37 56
8 16 51
3 7 48
0.6 2 33
0.2 1 86
0.2 1 89
assuming that this population is dynamic and that there are other institutions and also persons, not registered at the IBAMA, that kept these species in captivity. The most important reports on the occurrence of antibodies anti-Toxoplasma gondii in captive neotropical felids are summarized in Table 2. These data show that the infection is widely disseminated among neotropical felids kept in captivity in the Americas and suggest that the risk of exposure to T. gondii in these animals varies greatly from place to place. The total sero-prevalences for males and females were, respectively, 55% (95% CI: 52%, 58%) and 54% (95% CI: 51%, 57%), showing that there were no differences of susceptibility or risk of exposure between sexes. Table 3 shows that most of the animals were kept alone or in couples or groups of two animals of the same sex. It was not possible to carry out a logistic regression with random effects, technically the most suitable method in this case, because the computational algorithms did not converge due to the fact that many clusters had only one animal (Table 3). Thus, to estimate the risk factors, we used a logistic regression without random effects. Table 4 Distribution of risk factors for sero-positivity to T. gondii in 865 captive wild felids from 86 Brazilian zoos and breeding centers (1995–2001) Variable
Sero-positive
Sero-negative
Exposed
Total
Exposed
Total
Age >3 years
440
464
282
397
Animal origin Wild Born in the institution Another institution Donation Circus
126 81 92 46 3
464 494 464 464 464
97 112 72 30 1
368 398 398 398 398
Animal lived in a group Another animal was introduced in the group Time in the institution >1 year Eating meat previously frozen for a period <7 days in the last 6 months Eating meat of run-over or euthanized animals in the last 6 months Predation of rodents and birds in the last 6 months Presence of domestic cats near the enclosures in the last 6 months
279 136 270 386 271 90 248
426 458 320 439 442 442 442
238 91 183 295 160 57 192
366 397 270 378 383 383 383
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Table 5 Results of the multiple logistic regression analysis for risk factors associated with sero-positivity to T. gondii in 865 captive wild felids from 86 zoos and breeding centers from Brazil (1995–2001) Variable
b
S.E.(b)
OR
95% CI
P*
Age >3 years Eating meat previously frozen for a period <7 days Eating meat of run-over or euthanized animals
1.56 0.80 0.49
0.28 0.29 0.19
4.75 2.23 1.64
2.75, 8.2 1.24, 4.01 1.14, 2.37
0.001 0.007 0.008
Intercept = 2.12, P < 0.001; log likelihood statistic = 701.82; Hosmer–Lemeshow x2 = 1.075 (d.f. = 3, P = 0.78). * Wald’s test.
Table 4 shows the results of the univariable analysis and Table 5 shows the final model of the multiple logistic regression. The observed significance level for the Hosmer and Lemeshow goodness-of-fit Chisquare (x2 = 1.075, d.f. = 3) was 0.78, so the logistic regression model fitted the data reasonably well. Examining the variance-inflation factor (all values below 1.2), no collinearity was observed between the variables that passed screening but were not selected for the model and the risk factors in the final logistic model. It is reasonable to suppose that the greater the age, the greater the possibility of exposure to the risk of infection by T. gondii. However, the possibility of intervention on the variable ‘‘age’’, in a zoo, is null for the animals already housed. Knowledge that older cats are more likely to be infected might be used when planning management to other felids and animal keepers. It is interesting to us observe that although some variables related to the risk of exposure by means of the ingestion of oocysts were selected and submitted to multiple logistic regression (Table 4), none of them remained in the final model, indicating that the greatest risk of exposure is the ingestion of viable cysts in tissues. This is related to feed management, a variable in which intervention is possible. Thus, in the zoo, the exposure of neotropical felids to the T. gondii could be reduced by feeding the animals only with meat previously frozen at 12 8C for a period >7 days (Kotula et al., 1991). In Brazil, feeding captive felids with carcasses of runover or euthanized animals is an acceptable and very common practice. The implementation of this routine depends on the availability of equipment and storage capacity, which are possible to be arranged, but are not always found in some visited institutions. Considering that the elimination of oocysts has been shown in 5 out of 10 existing neotropical felids species (Jewell et al., 1972; Miller et al., 1972; Pizzi et al., 1978; Aramini et al., 1998; Lukesˇova´ and Litera´k, 1998), it is possible to state that after the intervention, a reduction in the load of oocysts eliminated in the environment is expected. The reduction of the oocysts environmental contamination is particularly interesting when felids are kept near by Australian marsupials and neotropical primates, which are species highly susceptible to toxoplasmosis (Dubey and Beattie, 1988; Garrel, 1999; Epiphanio et al., 2000).
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4. Conclusions The most effective way to reduce the risk of exposure of captive neotropical felids to T. gondii infection in Brazil and consequently reduce the oocysts environmental contamination is feeding these animals only with meat previously frozen at 12 8C for a period >7 days.
Acknowledgements The authors are grateful to the zoos and breeding centers staffs, IBAMA and Associac¸a˜o Mata Ciliar. They also thank Dr. J.P. Dubey (ARS, USDA) for laboratory support and CAPES and FAPESP for financial support.
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